1. Technical Field
The invention relates to methods and materials involved in the activation and inhibition of opiate, cannabinoid, and estrogen receptors. Specifically, the invention relates to mu3 opiate receptors, cannabinoid receptors, and estrogen surface receptors, and the biological responses induced by such receptors.
2. Background Information
Three general classes of cell surface opioid receptors (kappa, delta, and mu) have been described based on ligand specificity. Opioid receptors exhibiting high binding specificity for morphine have been designated mu opioid receptors. Detailed analysis of mu opioid receptors from various tissue has revealed the existence of multiple mu opioid receptor subtypes. In fact, the cDNA encoding the mu1 opioid receptor subtype has been identified. Oligonucleotides complementary to some, but not all, exons of the mu1 opioid receptor can block the effects mediated by the mu1 and mu2 receptor subtypes. Thus, the mu1 and mu2 opioid receptor subtypes appear to share exon sequences, as would be expected of splice variants. Supporting the idea of alternative splicing is the finding of a single mu gene in mouse chromosomal DNA. In addition, a novel rat brain mu opioid receptor subtype, designated rMOR1B, has been identified. This receptor is identical to the rat mu1 opioid receptor at its N-terminus but differs in its length and sequence at the C-terminus. Further, affinity studies demonstrated that the substrate specificity of rMOR1B is similar to that of the rat mu1 opioid receptor, but rMOR1B is more resistant to agonist-induced desensitization and has a different expression pattern in brain. The presence of another opiate receptor, designated mu3 opiate receptor, has been demonstrated pharmacologically. This mu3 opiate receptor is opioid peptide insensitive and opiate alkaloid selective. In addition, detailed binding analysis indicates that the mu3 opiate receptor is expressed by immune tissues (e.g., human monocytes and granulocytes).
Cannabinoids induce physiological activities similar to those induced by morphine. Cannabinoids, however, induce these activities by interacting with specific cannabinoid receptors that are structurally distinct from opioid receptors. To date, two subtypes of G-protein-coupled cannabinoid receptors have been identified: CB1 and CB2. These two cannabinoid receptor subtypes are expressed by different tissues and have different pharmacological properties. For example, the CB1 receptor is expressed in brain and endothelial tissue while the CB2 receptor is expressed in the immune system. In addition, SR 141716A is a CB1 receptor antagonist while SR 144528 is a CB2 receptor antagonist.
Estrogen, like morphine, appears to have multiple receptors. In fact, two different cDNA clones have been identified: one encoding estrogen receptor-alpha (ER-alpha) and the other encoding estrogen receptor-beta (ER-beta). In addition, many variants of ER-beta have been described, including human ER-beta isotypes 1 through 5. Unlike the mu opioid receptor subtypes, however, these estrogen receptors are intracellular nuclear receptors. Presumably, each of these intracellular estrogen receptors, upon interaction with estrogen, mediate biological responses by interacting directly with DNA. Tamoxifen is a lipophilic anti-estrogen compound that can inhibit the interaction of estrogen with intracellular nuclear receptors.
The present invention relates generally to mu3 opiate receptors, cannabinoid receptors, and estrogen surface receptors (ESRs). Specifically, the invention provides methods and materials for identifying mu3 opiate receptor agonists and antagonists, cannabinoid receptor agonists and antagonists, and ESR agonists and antagonists. In addition, the invention provides an isolated nucleic acid molecule that encodes a mu3 opioid receptor, a host cell containing an isolated nucleic acid molecule that encodes a mu3 opioid receptor, and an isolated mu3 opioid receptor polypeptide. Further, the invention provides methods and materials for treating cancers, inflammatory conditions, sepsis conditions, viral infections, and cardiovascular diseases.
The present invention is based on the discovery of a cell surface receptor for estrogen. Specifically, this cell surface receptor for estrogen, designated estrogen surface receptor 1 (ESR1), exhibits ligand specificity for estrogen and 17xcex2-estradiol (E2) as well as E2 conjugated to bovine serum albumen (E2-BSA). In addition, ESR1 is tamoxifen sensitive. In other words, tamoxifen can inhibit the stimulatory effects of ESR1 agonists such as estrogen and E2. Thus, tamoxifen is an ESR1 antagonist. Further, ESR1 is expressed by human endothelial cells. Moreover, the ESR1-mediated biological responses induced by ESR1 agonists include changes in intracellular calcium concentration and nitric oxide release. The existence of a cell surface receptor for estrogen has significant medical implications. For example, plasma membrane impermeable compounds can be used to influence the biological effects of estrogen. Taken together, the discovery of ESR1 and the biological responses mediated by ESR1 provides methods and materials for modulating calcium and nitric oxide regulated mechanisms. As described herein, modulating calcium and nitric oxide regulated mechanisms can be used to treat cancers, inflammatory conditions, sepsis conditions, viral infections, and cardiovascular diseases.
The present invention is also based on the discovery of several assays for identifying mu3 opiate receptor, cannabinoid receptor, or ESR agonists and antagonists. Specifically, the assays involve monitoring at least one biological response induced by mu3 opiate receptors, cannabinoid receptors, or ESRs. Such biological responses include changes in intracellular calcium concentration and nitric oxide release. Thus, the assays can be configured to monitor intracellular calcium concentration, nitric oxide release, or both. The assays are particularly advantageous since the biological responses induced by mu3 opiate receptors, cannabinoid receptors, or ESRs can be detected within seconds of applying an agonist. Thus, many test molecules can be screened rapidly for the ability to either stimulate or inhibit mu3 opiate receptor, cannabinoid receptor, or ESR activities. In addition, the assays are specific for the particular receptor subtype. For example, stimulation or inhibition of mu3 opiate receptor activity can be easily distinguished from effects operating through mu1 or mu2 opioid receptors. Likewise, stimulation or inhibition of CB1 activity can be easily distinguished from effects operating through CB2, and stimulation or inhibition of ESR activity can be easily distinguished from effects operating through the intracellular nuclear receptors for estrogen (e.g., ER-alpha and ER-beta). Further, any results generated from an assay that, for example, monitors intracellular calcium concentration can be easily confirmed by performing an assay that monitors nitric oxide release. Having the ability to confirm a particular test molecule""s ability to stimulate or inhibit mu3 opiate receptor, cannabinoid receptor, or ESR activity provides a powerful tool for reliably identifying receptor agonists and antagonists. Taken together, the assays described herein can be used to identify mu3 opiate receptor, cannabinoid receptor, or ESR agonists and antagonists rapidly, specifically, and reliably.
In addition, the present invention is based on the discovery of an isolated nucleic acid molecule that encodes a mu3 opiate receptor. Specifically, the mu3 opiate receptor is a cell surface receptor that exhibits specificity for morphine while being opioid peptide insensitive. In addition, the interaction of morphine with the mu3 opiate receptor induces changes in intracellular calcium concentration and nitric oxide release. Isolated nucleic acid molecules that encode the mu3 opioid receptor, isolated mu3 opioid receptor polypeptides, and host cells containing such isolated nucleic acid molecules are particularly useful to research scientists since these materials allow scientists to explore, for example, the interactions of morphine with the mu3 opiate receptor, the molecular mechanisms by which morphine binding induces intracellular calcium concentration changes, and the relationships of the mu3 opiate receptor with other mu opioid receptors. In addition, these materials can be used to transform mu3 opiate receptor negative cells into mu3 opiate receptor positive cells.
Further, the present invention is based on the discovery of relationships between mu3 opiate receptor, cannabinoid receptor, and ESR activities and various disease conditions. Specifically, disease conditions such as cancer can be treated with specific mu3 opiate receptor, cannabinoid receptor, or ESR antagonists, either individually or in various combinations. In addition, disease conditions such as inflammatory conditions, sepsis conditions, viral infections, and cardiovascular diseases can be treated with specific mu3 opiate receptor, cannabinoid receptor, or ESR agonists, either individually or in various combinations.
In general, the invention features a method for identifying a mu3 opiate receptor agonist. This method involves contacting a cell (e.g., cancer cell) with a test molecule and determining if the test molecule induces a mu3 opiate receptor-mediated response in the cell in a mu3 opiate receptor-specific manner. The cell expresses a mu3 opiate receptor (e.g., a human mu3 opiate receptor) and the test molecule is a molecule other than morphine or dihydromorphine. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
In another embodiment, the invention features a method for identifying a mu3 opiate receptor antagonist. The method involves contacting a cell (e.g., cancer cell) with a test molecule and a mu3 opiate receptor agonist (e.g., morphine or dihydromorphine), and determining if the test molecule influences (e.g., reduces) induction of a mu3 opiate receptor-mediated response in the cell by the mu3 opiate receptor agonist. The cell expresses a mu3 opiate receptor, and the test molecule is a molecule other than naloxone or naltrexone. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
Another embodiment of the invention features a method for identifying a cannabinoid receptor agonist. The method involves contacting a cell (e.g., endothelial cell) with a test molecule, and determining if the test molecule induces a cannabinoid receptor-mediated response in the cell in a cannabinoid receptor-specific manner. The cell expresses a cannabinoid receptor (e.g., a human cannabinoid receptor such as CB1), and the test molecule is a molecule other than anandamide. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
Another embodiment of the invention features a method for identifying a cannabinoid receptor antagonist. The method involves contacting a cell (e.g., endothelial cell) with a test molecule and a cannabinoid receptor agonist (e.g., anandamide), and determining if the test molecule influences (e.g., reduces) induction of a cannabinoid receptor-mediated response in the cell by said cannabinoid receptor agonist. The cell expresses a cannabinoid receptor (e.g., CB1 receptor), and the test molecule is a molecule other than SR 141716A. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
Another embodiment of the invention features a method for identifying an estrogen surface receptor agonist. The method involves contacting a cell (e.g., endothelial cell) with a test molecule, and determining if the test molecule induces an estrogen surface receptor-mediated response in the cell in an estrogen surface receptor-specific manner. The cell expresses an estrogen surface receptor such as a human estrogen surface receptor. The estrogen surface receptor can be ESR1. The test molecule can be plasma membrane impermeable. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
Another embodiment of the invention features a method for identifying an estrogen surface receptor antagonist. The method involves contacting a cell with a test molecule and an estrogen surface receptor agonist, and determining if the test molecule influences (e.g., reduces) induction of an estrogen surface receptor-mediated response in the cell by the estrogen surface receptor agonist. The cell expresses an estrogen surface receptor such as a human estrogen surface receptor. The estrogen surface receptor can be ESR1. The test molecule can be plasma membrane impermeable. The estrogen surface receptor agonist can be estrogen, 17xcex2-estradiol, or 17xcex2-estradiol-BSA. The determining step can involve monitoring nitric oxide synthase activity in the cell. For example, nitric oxide synthase activity can be monitored by detecting nitric oxide release from the cell. A nitric oxide-specific amperometric probe can be used to detect the nitric oxide release. The determining step can involve monitoring intracellular calcium levels within the cell. For example, a fluorescent ion indicator (e.g., Fura-2) can be used to monitor the intracellular calcium levels. In addition, the determining step can involve monitoring both nitric oxide synthase activity and intracellular calcium levels in the cell.
In another aspect, the invention features an isolated nucleic acid molecule having first and second nucleic acid sequences. The first nucleic acid sequence is substantially homologous to SEQ ID NO:1, and the second nucleic acid sequence is substantially homologous to SEQ ID NO:2. The first and second nucleic acid sequences are separated by more than about 1500 nucleotides. The isolated nucleic acid molecule can encode a mu3 opiate receptor polypeptide. In addition, the isolated nucleic acid molecule can have a third nucleic acid sequence at least about 80 percent identical to SEQ ID NO:5. The third nucleic acid sequence is located between the first and second nucleic acid sequences.
Another aspect of the invention features a host cell containing an isolated nucleic acid molecule. The isolated nucleic acid molecule has a first and second nucleic acid sequence. The first nucleic acid sequence is substantially homologous to SEQ ID NO:1, and the second nucleic acid sequence is substantially homologous to SEQ ID NO:2. The first and second nucleic acid sequences are separated by more than about 1500 nucleotides.
Another aspect of the invention features an isolated polypeptide having an amino acid sequence at least about 80 percent identical to SEQ ID NO:6. The polypeptide is between 403 and 600 amino acid residues.
Another aspect of the invention features a method for treating a mammal (e.g., human) having cancer. The method involves administering a mu3 opiate receptor antagonist to the mammal such that a mu3 opiate receptor-mediated response is reduced. The reduction of the mu3 opiate receptor-mediated response promotes anti-tumor activity in the mammal. The cancer can be lung cancer, breast cancer, prostate cancer, colon cancer, carcinoma, leukemia, or melanoma. The mu3 opiate receptor-mediated response can be a change in intracellular calcium concentration in a cell (e.g., cancer cell) within the mammal. The mu3 opiate receptor-mediated response can be a change in the amount of nitric oxide released from a cell within the mammal. The method can involve administering a cannabinoid receptor antagonist (e.g., a CB1 receptor antagonist) to the mammal such that a cannabinoid receptor-mediated response is reduced. The method can involve administering an estrogen surface receptor antagonist (e.g., an ESR1 antagonist) to the mammnal such that an estrogen surface receptor-mediated response is reduced. The estrogen surface receptor antagonist can be plasma membrane impermeable, for example, tamoxifen coupled to bovine serum albumin.
In another embodiment, the invention features a method for treating a mammal having cancer. The method involves administering a cannabinoid receptor. antagonist (e.g., a CB1 receptor antagonist) to the mammal such that a cannabinoid receptor-mediated response is reduced. The reduction of the cannabinoid receptor-mediated response promotes anti-tumor activity in the mammal.
Another embodiment of the invention features a method for treating a mammal having cancer. The method involves administering an estrogen surface receptor antagonist (e.g., an ESR1 antagonist) to the mammal such that an estrogen surface receptor-mediated response is reduced. The estrogen surface receptor antagonist is plasma membrane impermeable. In addition, the reduction of the estrogen surface receptor-mediated response promotes anti-tumor activity in the mammal.
Another embodiment of the invention features a method for treating a mammal having an inflammatory condition. The method involves administering a mu3 opiate receptor agonist to the mammal such that a mu3 opiate receptor-mediated response is induced. In addition, the induction of the mu3 opiate receptor-mediated response promotes anti-inflammatory or immunosuppressive activity in the mammal. The inflammatory condition can be arthritis, pericarditis, vasculitis, lupus, bronchitis, or phrenitis. The method can involve administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The method can involve administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced.
Another embodiment of the invention features a method for treating a mammal having an inflammatory condition. The method involves administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The induction of the cannabinoid receptor-mediated response promotes anti-inflammatory or immunosuppressive activity in the mammal.
Another embodiment of the invention features a method for treating a mammal having an inflammatory condition. The method involves administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced. The induction of the estrogen surface receptor-mediated response promotes anti-inflammatory or immunosuppressive activity in the mammal.
Another embodiment of the invention features a method for treating a mammal having sepsis. The method involves administering a mu3 opiate receptor agonist to the mammal such that a mu3 opiate receptor-mediated response is induced. The induction of the mu3 opiate receptor-mediated response reduces septic shock in the mammal. The method can involve administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The method can involve administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced.
Another embodiment of the invention features a method for treating a mammal having sepsis. The method involves administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The induction of the cannabinoid receptor-mediated response reduces septic shock in the mammal.
Another embodiment of the invention features a method for treating a mammal having sepsis. The method involves administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced. The induction of the estrogen surface receptor-mediated response reduces septic shock in the mammal.
Another embodiment of the invention features a method for treating a mammal having a viral infection (e.g., HIV infection). The method involves administering a mu3 opiate receptor agonist to the mammal such that a mu3 opiate receptor-mediated response is induced. The induction of the mu3 opiate receptor-mediated response promotes an anti-viral response in the mammal. The method can involve administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The method can involve administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced.
Another embodiment of the invention features a method for treating a mammal having a viral infection. The method involves administering a cannabinoid receptor agonist to the mammal such that a cannabinoid receptor-mediated response is induced. The induction of the cannabinoid receptor-mediated response promotes an anti-viral response in the mammal.
Another embodiment of the invention features a method for treating a mammal having a viral infection. The method involves administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced. The induction of the estrogen surface receptor-mediated response promotes an anti-viral response in the mammal.
Another embodiment of the invention features a method for treating a mammal having cardiovascular disease. The method involves administering an estrogen surface receptor agonist to the mammal such that an estrogen surface receptor-mediated response is induced. The estrogen surface receptor agonist is plasma membrane impermeable. The induction of the estrogen surface receptor-mediated response can reduce or prevent atherosclerosis in the mammal.
Another embodiment of the invention features a method for treating a mammal with a mu3 opiate receptor agonist such that mu3 opiate receptor-mediated nitric oxide release is suppressed. The method involves administering an opioid receptor agonist to the mammal followed by administering the mu3 opiate receptor agonist. The opioid receptor agonist is not a mu3 opiate receptor agonist. The opioid receptor agonist can be DAMA, xcex2-endorphin, or DAMGO. The mu3 opiate receptor agonist can be morphine or dihydromorphine.
Another aspect of the invention features a pharmaceutical formulation containing a mu3 opiate receptor antagonist and an estrogen surface receptor antagonist.
In another embodiment, the invention features a pharmaceutical formulation containing a mu3 opiate receptor antagonist and a cannabinoid receptor antagonist.
Another embodiment of the invention features a pharmaceutical formulation containing an estrogen surface receptor antagonist and a cannabinoid receptor antagonist.
Another embodiment of the invention features a pharmaceutical formulation containing an estrogen surface receptor antagonist that is membrane impermeable.
Another embodiment of the invention features a pharmaceutical formulation containing a mu3 opiate receptor agonist and an estrogen surface receptor agonist.
Another embodiment of the invention features a pharmaceutical formulation containing a cannabinoid receptor agonist and an estrogen surface receptor agonist.
Another aspect of the invention features the use of a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor antagonist in the manufacture of a medicament for the treatment of cancer. The estrogen surface receptor antagonist is plasma membrane impermeable.
In another embodiment, the invention features the use of a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition.
Another embodiment of the invention features the use of a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist in the manufacture of a medicament for the treatment of sepsis.
Another embodiment of the invention features the use of a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist in the manufacture of a medicament for the treatment of a viral infection.
Another embodiment of the invention features the use of an estrogen surface receptor agonist in the manufacture of a medicament for the treatment of cardiovascular disease. The estrogen surface receptor agonist is plasma membrane impermeable.
Another aspect of the invention features an article of manufacture containing packaging material and a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor antagonist contained within the packaging material. The packaging material contains a label or package insert indicating that the mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor antagonist can be administered to a mammal to treat cancer. The estrogen surface receptor antagonist is plasma membrane impermeable.
In another embodiment, the invention features an article of manufacture containing packaging material and a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist contained within the packaging material. The packaging material contains a label or package insert indicating that the mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist can be administered to a mammal to treat an inflammatory condition.
Another embodiment of the invention features an article of manufacture containing packaging material and a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist contained within the packaging material. The packaging material contains a label or package insert indicating that the mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist can be administered to a mammal to reduce septic shock.
Another embodiment of the invention features an article of manufacture containing packaging material and a mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist contained within the packaging material. The packaging material contains a label or package insert indicating that the mu3 opiate receptor, cannabinoid receptor, or estrogen surface receptor agonist can be administered to a mammal to treat a viral infection.
Another embodiment of the invention features an article of manufacture containing packaging material and an estrogen surface receptor agonist contained within the packaging material. The packaging material contains a label or package insert indicating that the estrogen surface receptor agonist can be administered to a mammal to treat cardiovascular disease. The estrogen surface receptor agonist is plasma membrane impermeable.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.